Modulation of immune responses against CNS antigens, such as myelin basic protein (MBP) and myelin oligodendrocyte glycoprotein (MOG), is a goal for therapeutic intervention in multiple sclerosis. The focus of our lab has been to develop novel approaches for the induction of antigen-specific tolerance that can be applied to the prevention or reversal of undesirable immune responses, e.g., in autoimmune diseases like MS. During the last decade, we created a platform technology that can be used for gene therapy to induce tolerance to multiple epitopes. In our model, LPS-activated B cells are transduced via retroviral vectors to express an Ig fusion protein with the targeted epitopes. Data in four experimental autoimmune models have demonstrated clinical efficacy in that expression of Ig fusion proteins of autoantigens by B cells can both prevent and reverse autoimmune responsiveness. Importantly, we found that CD25+, FoxP3+ regulatory T cells were required for both the induction and maintenance of tolerance in this system. We also reported that the mode of activation of the transduced B cells was a critical factor for the success of B cell-delivered gene therapy for tolerance, an observation that is important for potential translation. We hypothesize that different B cell activators lead to the production of distinct sets of chemokines and cytokines and/or the stimulation of tolerogenic (marginal zone) versus non-tolerogenic B cells. Our hypothesis is that transduced antigen-presenting B cells (APC), in particular marginal zone B cells, present the processed epitopes to effector T cells and/or Tregs to inhibit responsiveness via regulatory cytokines. Further, we suggest that the tolerogenic B cells directly recruit Tregs via CTLA-4 to induce tolerance. In order to test these hypotheses and to translate our efforts to the clinic for potential future treatment of MS, we will focus in this renewal on the following three specific aims. (1) Based on the observation that LPS- and CpG-activated B cells produce different cytokines and chemokines, using knockout mice, we will define the roles of specific soluble mediators generated under these and other activation conditions. We will also analyze the lifespan and phenotype of transduced B cells, as well as their migration to lymphoid tissues and the CNS. We will test the hypothesis that anti-CD20 treatments leads to partial depletion of B cells, sparing the tolerogenic MZ cells. (2) We will determine the migration and fate of the TCR transgenic T cells in B cell delivered gene therapy, and will analyze the kinetics of their interaction with tolerogenic or non-tolerogenic B cells using two photon microscopy. (3) In our final aim, we will use humanized systems to begin to translate these studies to human disease by analyzing responses of HLA DR2 transgenic mice and of human T cells in vitro, including the activation of Tregs by gene therapy. We will also examine the efficacy of non-integrating vectors (e.g., gutless adenovirus) for B-cell delivered gene therapy in both the later system and in EAE models. These studies will provide proof of principle with human T cells in a clinically relevant model, will establish the mechanisms of B cell-delivered gene therapy for tolerance, and will move this project forward to translation in the clinic as a potential therapy for multiple sclerosis. PUBLIC HEALTH RELEVANCE T cells from multiple sclerosis patients have been shown to respond to a variety of CNS antigens, such as myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) and phospholipoprotein (PLP); hence, modulation of these immune responses is a goal for therapeutic intervention in MS. The focus of our lab has been to develop a novel B-cell delivered gene therapy method for the induction of tolerance, particularly in autoimmune diseases like MS. This platform has been successfully applied in murine models for MS. Herein, we will investigate the mechanism of B-cell delivered tolerance in animal models, and then we will begin to translate these studies to the clinic using T cells from MS patients exposed to B cells transduced with MBP-Ig constructs in vitro. These studies will establish a novel approach for tolerance with human T cells in a clinically relevant model, and will continue the translation of this project forward to the clinic as a potential therapy for multiple sclerosis.